一种基于重组的方法，用于快速评估酵母双杂交克隆的特异性。
A recombination based method to rapidly assess specificity of two-hybrid clones in yeast.
作者信息
Petermann R, Mossier B M, Aryee D N, Kovar H
机构信息
Children's Cancer Research Institute, St. Anna Kinderspital, Kinderspitalgasse 6, A-1090 Vienna, Austria.
出版信息
Nucleic Acids Res. 1998 May 1;26(9):2252-3. doi: 10.1093/nar/26.9.2252.
Abstract
The yeast two-hybrid system is frequently used to identify protein-protein interactions. Confirming the specificity of candidate clones requires separation and isolation of yeast plasmids, propagation in bacteria and testing combinations of DNA-binding and activation domain hybrids in yeast. In order to simplify this procedure, we developed a rapid method based on PCR amplification of library insert DNAs and in vivo cloning into the activation domain hybrid vector. Reporter gene activity is assayed in parallel for combinations with different DNA-binding domain hybrids. Further characterization of inserts does not require plasmid isolation and intermediate hosts.
摘要
酵母双杂交系统常用于鉴定蛋白质-蛋白质相互作用。确认候选克隆的特异性需要分离和纯化酵母质粒，在细菌中扩增并在酵母中测试DNA结合结构域和激活结构域杂交体的组合。为了简化这一过程，我们开发了一种基于PCR扩增文库插入DNA并在体内克隆到激活结构域杂交载体的快速方法。同时检测与不同DNA结合结构域杂交体组合的报告基因活性。对插入片段的进一步鉴定不需要质粒分离和中间宿主。
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